Fish IKKα from Japanese eel (Anguilla japonica) can activate NF-κB, AP1, and type I IFN signaling pathways

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKK alpha) plays a pivotal role in the activation of nuclear factor kappa-B (NF-kappa B) pathway in response to pathogens infections in mammals, but the information about IKK alpha in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKK alpha homologue, AjIKK alpha, was cloned by 5' and 3' SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKK alpha GFP fusion protein and the induction of AjIKKa in the activation of NF-kappa B, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKK alpha has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKK alpha is similar to that of human IKK alpha. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKK alpha in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKK alpha expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKK alpha in the spleen. In vitro, the AjIKK alpha transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 x 10(6) cfu/mL, 1 x 10(7) cfu/mL, and 1 x 108 cfu/mL). Luciferase assays demonstrated that AjIKK alpha expression could significantly induce NF-kappa B, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKK alpha was evenly distributed in the cytoplasm in the natural state, but AjIKK alpha was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKK alpha plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-kappa B, AP1and type I IFN signaling pathway.